Abstract

Macrophages are critical effectors of bacterial clearance and must retain viability, despite exposure to toxic bacterial products, until key antimicrobial functions are performed. Subsequently, host-mediated macrophage apoptosis aids resolution of infection. The ability of macrophages to make this transition from resistance to susceptibility to apoptosis is important for effective host innate immune responses. We investigated the role of Mcl-1, an essential regulator of macrophage lifespan, in this switch from viability to apoptosis, using the model of pneumococcal-associated macrophage apoptosis. Upon exposure to pneumococci, macrophages initially upregulate Mcl-1 protein and maintain viability for up to 14 hours. Subsequently, macrophages reduce expression of full-length Mcl-1 and upregulate a 34-kDa isoform of Mcl-1 corresponding to a novel BH3-only splice variant, Mcl-1Exon-1. Change in expression of Mcl-1 protein is associated with mitochondrial membrane permeabilization, which is characterized by loss of mitochondrial inner transmembrane potential and translocation of cytochrome c and apoptosis-inducing factor. Following pneumococcal infection, macrophages expressing full-length human Mcl-1 as a transgene exhibit a delay in apoptosis and in bacterial killing. Mcl-1 transgenic mice clear pneumococci from the lung less efficiently than nontransgenic mice. Dynamic changes in Mcl-1 expression determine macrophage viability as well as antibacterial host defense.

Figure 1

Mcl-1 protein levels demonstrate biphasic variation in association with pneumococcal infection of macrophages. (A) Total RNA was extracted from MDMs 20 hours after mock infection (Spn–) or infection with pneumococci (Spn+), and Mcl-1 mRNA was detected by RNase protection assay. The results presented are typical of 5 donors tested, but the duration of mRNA elevation varied among donors. L32 and GAPDH served as loading controls. (B) Western blot of total protein from MDMs at the indicated time points after infection, probed with anti–Mcl-1 and anti-actin antibodies. Data are representative of 7 independent experiments. (C) Mcl-1 band density from a series of MDMs as in B, quantified by densitometry (n = 7). P < 0.05, Mcl-1 at 6 hours vs. 12 hours; P < 0.001, 6 hours vs. 20 hours; Student’s t test. (D) Levels of nuclear apoptosis in the same series of MDMs as in C. P < 0.001, apoptosis at 6 hours vs. 16 hours, Student’s t test.